Methods for increasing palatability of pet foodstuff

ABSTRACT

The present invention relates to a method of identifying a compound that binds to or modulates the activity of one or more polypeptides encoding one or more receptors that are involved in the detection and perception of fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/246,091, which is a divisional of U.S. patent application Ser. No.14/898,321, filed Dec. 14, 2015, now U.S. Pat. No. 10,222,387, which isa U.S. National Stage patent application under 35 U.S.C. § 371 of andclaims priority to International Application No. PCT/GB2014/000233,filed Jun. 13, 2014, which claims priority to GB Patent Application No.1310664.6, filed Jun. 14, 2013, the contents of each of which are herebyincorporated by reference in their entireties, and to which priority isclaimed.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Pursuant to 37 C.F.R. § 1.52(e)(5), theSequence Listing text file, identified as069269_0295CON_Sequence_Listing.txt, is 40,982 bytes and was created onAug. 8, 2019. The Sequence Listing, electronically filed herewith, doesnot extend beyond the scope of the specification and thus does notcontain new matter.

FIELD

The present invention relates to a method of identifying a compound thatbinds to or modulates the activity of one or more polypeptides encodingone or more receptors that are involved in the detection and perceptionof fatty acids.

BACKGROUND OF THE INVENTION

It is well known that many feline and canine companion animals are fussywith their food. An animal will often refuse to eat a foodstuff that ithas been eating for some time, or refuse to eat any more than a minimalamount of a foodstuff. Part of this phenomenon can be driven by subtledifferences in the sensory profile of the raw materials. Thesedifferences might not be perceived by the human consumer, but due todifferences in the olfactory and gustatory systems, feline and caninecompanion animals may well perceive these differences. These sensorydifferences can be due to natural variation of the raw materials used orwhen materials are in short supply and have to be substituted withalternatives. This can be very frustrating for the owner and can resultin the owner perceiving that the animal is unhappy and not enjoying itsfood. An animal may also fail to ingest its required amount of essentialnutrients if not consuming an adequate amount of food available to it.Therefore, it can clearly be seen that there exists a need for a way toencourage companion animals to eat the foodstuff with which it isprovided. Many solutions have been suggested to overcome this problem.Most commercially available pet foods are provided in a range ofdifferent flavours and/or textures. However, the companion animal ownerwill know that often a companion animal will suddenly, for no clearreason, refuse the flavour that the owner perceives to be its mostpreferred. Much research has been carried out on the flavour preferencesof companion animals, by offering them a choice of different foodstuffs.

Taste perception in mammalian animals is governed by the taste receptorsfound on taste buds of the tongue of the animal and has generally beenconsidered to involve five taste perceptions; salt, sweet, bitter, sourand umami. The taste of a food is determined by which receptors arestimulated. Although some taste receptors share homology betweenspecies, the prevalence, frequency and activity of each receptor typedepends on the species, since, as would be expected, an herbivorousanimal will require different taste stimuli than a carnivorous animal.Feline and canine taste receptors share some homology with those ofhuman, although, as is known, different receptors have different levelsof activation and/or preference in feline and canine animals than inhumans.

The perception of fat in foods is generally thought to have been due tomouth feel and, to some extent, smell. However, in the human and rodentfields, fatty acid taste receptors have recently been identified(Cartoni et al, 2010; Galindo et al 2012; Martin et al, 2011),indicating that a taste response is also involved in fat perception anddetection.

GPR120 (also known as GPR129, 03FAR1, PGR4, FFAR4) is predicted to be aG-protein coupled cell surface receptor, containing seven transmembranedomains (as well as an extracellular portion) involved in the detectionof specific fatty acids, and the G-protein associated intracellularportion involved in signal transduction. GPR120 is thought to bindmedium to long-chain fatty acids, such as oleic acid and linoleic acid,in their free form. It has been predicted that two isoforms (splicevariants) of the GP120 receptor exists in humans, GPR120L and GPR120S,on colonic endocrine cells. It has been suggested that the long isoformdoes not signal functionally in the perception of taste.

GPR120 is expressed in various mammalian tissue, and has been known tobe involved in the stimulation of cholecystokinin (CCK) secretion fromSTC-1 an intestinal secretory cell line, in addition it has beenreported that GPR120 has stimulatory effects on the secretion ofglucogon-like peptide (GLP-1). GPR120 is also expressed in the pituitarygland and therefore its potential involvement in stress regulation hasalso been explored. GPR120 is a known receptor for unsaturated longchain fatty acids and is involved in GLP-1 secretion, insulinsensitisation and anti-inflammatory and anti-obesity effects. It hasbeen suggested that GPR120 agonists or antagonists could be useful aspotential therapeutics for the treatment of various metabolic diseases,such as diabetes. However, GPR120 has yet to be explored for itspotential palatability enhancing effects.

EP 1688138A1 (Takeda Pharmaceutical Company Limited) is a Europeanpatent application directed towards a specific agent for regulatinghuman derived 14273 receptor (GPR120 receptor) function. The documentdescribes low molecular weight synthetic agonists or antagonists forstimulating GPR120. These substances are stated to be useful for thetreatment of over-eating, diabetes, or obesity. Alternative agentscapable of suppressing GPR120 are described and their use in thetreatment of anorexia. The application is limited to human and mouseGPR120 receptors and relates to the identification and use of compoundsin a therapeutic context.

Patent application publication WO 2007/134613 (Rheo-Science A/S) relatesto GPR120 receptor expression in various mammalian tissues. Theapplication suggests the use of a compound for modulating the expressionof GPR120 in order to treat, alleviate, prevent or diagnose diabetesand/or obesity i.e. therapeutic applications in humans.

European patent application EP 1932920A1 (Eisai R&D Management Co Ltd)discloses a method for determining whether a substance alters humanGPR120 mediated cell stimulating activity for therapeutic applications.

Patent application publication WO 2011/159297A1 (Metabolex Inc)describes human and rat GPR120 agonists and their use in the treatmentof metabolic diseases including diabetes and diseases associated withpoor glycaemic control. This application describes that GPR120 agonistswere administered to mice to determine the effects on secretion ofinsulin, glucogon-like peptide 1 and various other hormones. It wasshown that GPR120 agonists can lower blood glucose in response to anintra peritoneal glucose challenge in mice.

Bharat Shimpukade et al (Journal of Medicinal Chemistry, Discovery of aPatent and Selective GPR120 Agonist, 2012 May 10; 59(9):4511-4515)disclose a human GPR120 agonist for therapeutic use.

Qi Sun et al (Molecular Pharmacology, Structure-Activity Relationshipsof GPR120 Agonists Based on a Docking Simulation, 2010 November;78(5):804-810) describe human GPR120 agonists for therapeutic purposes.

Takafumi Hara et al (Naunyn-Schmied Arch Pharmacol, Novel SelectiveLigands for Free Fatty Acid Receptors GPR120 and GPR40, 2009 September;380(3):247-255) attempt to identify new therapeutic ligands for humanGPR120 receptor. However, the authors were only able to identify partialagonists.

Takayoshi Suzuki et al (Journal of Medicinal Chemistry, Identificationof G Protein-Coupled Receptor 120-Selective Agonists Derived from PPARγAgonists, 2008 Dec. 11; 51(23):7640-7644) describe the need to discoverGPR120 selective agonists as they can be used as therapeutic agents.

CD36 (also known a FAT, GP3B, GP4, GPIV, SCARB3, thrombospondinreceptor) does not belong to the G-protein coupled receptor family (itbelongs to the class B scavenger receptor family), which is unusual withreference to other known fatty acid taste receptors in humans.

Domestic feline animals are known to be fussy with food, and many ownersperceive that the cat will only eat certain food stuffs on certain days.Therefore, the ability to ensure that a cat responds well to aparticular foodstuff would ensure the consistent acceptance of afoodstuff by an animal, and also to ensure that the owner perceives thatthe animal is happy and healthy.

Canine animals can also be fussy or in the case of some animals,indiscriminate in food selection. By improving the taste perception offoodstuff, canine animals can be encouraged to eat a particularfoodstuff more reliably and consistently.

Currently, cats' and dogs' preference for taste stimuli are identifiedthrough feeding tests, which can be inefficient in terms of cost, timeand results. Furthermore, the identification of novel taste stimuli isdifficult, as many compounds may need to be tested and worked throughusing animal preference tests, in order to determine which may bereliably attractive to the feline and canine animals. Relatively largeamounts of each test compound are necessary for such methods.

Therefore, there is a need for reliable, more efficient screeningmethods for identifying taste compounds that can bind to and stimulate(or otherwise modulate) certain taste receptors in animals, canine andfeline animals in particular.

BRIEF SUMMARY OF THE INVENTION

The presently disclosed subject matter provides a method for identifyinga compound that binds to and/or modulates the activity of a polypeptidecomprising; (i) the sequence of a feline or canine GPR120 or a feline orcanine CD36 receptor; (ii) the amino acid sequence as set out in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7; (iii) an amino acidsequence having at least 90% identity to SEQ ID NO:1 or SEQ ID NO:3;(iv) an amino acid sequence having at least 90% identity to SEQ ID NO:5or SEQ ID NO:7; (v) an amino acid sequence comprising amino acids 127 to279 of SEQ ID NO:3 or SEQ ID NO:7; (vi) a functional fragment of (i),(ii), (iii), (iv) or (v). the method comprising determining whether atest compound binds to and/or modulates the activity of the polypeptide.

The presently disclosed subject matter further provides a method foridentifying a taste compound that binds to and/or modulates the activityof (a) GPR120 receptor and/or a CD36 receptor, wherein: a) the GPR120receptor is: i) feline, canine or human; ii) has the amino acid sequenceof SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9; or iii) is at least 87%identical to SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9; and b) the CD36receptor is: i) feline, canine or human; ii) has the amino acid sequenceof SEQ ID NO:3, 7 or 11; iii) is at least 83% identical to SEQ ID NO: 3,7 or 11.

In certain embodiments, the method is an in vitro method. In certainembodiments, the method is an in silico method.

In certain embodiments, the in vitro method comprises: (i) measuring thebiological activity of the polypeptide in the absence and in thepresence of a compound; and (ii) identifying a compound as one whichbinds to or modulates the biological activity of the polypeptide, whenthere is a difference between the biological activity in the absence,compared to the presence of the compound. In certain embodiments, the invitro method further comprises contacting the polypeptide with acompound.

In certain embodiments, the in silico method comprises: (a) predictingthe 3-dimensional (3D) structure of the polypeptide; (b) screening thepredicted 3D structure of the polypeptide in silico with a 3D structureof a test compound; (c) determining if the test compound fits a bindingsite of the polypeptide; and (d) identifying a compound as one whichbinds to and modulates the biological activity of the polypeptide, whenthe 3D structure of the compound fits the binding site of the 3Dstructure of the polypeptide.

The presently disclosed subject matter further provides a foodstuffcomprising an agent or compound identified by the method of anyone ofthe methods disclosed herein.

The presently disclosed subject matter further provides a kit fordetermining whether a compound or agent activates a polypeptidecomprising; (i) the sequence of a feline or canine GPR120 or a feline orcanine CD36 receptor; (ii) the amino acid sequence as set out in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7; (iii) an amino acidsequence having at least 90% identity to SEQ ID NO:1 or SEQ ID NO:3;(iv) an amino acid sequence having at least 90% identity to SEQ ID NO: 5or SEQ ID NO:7; (v) an amino acid sequence comprising amino acids 127 to279 of SEQ ID NO:3 or SEQ ID NO:7; (vi) a functional fragment of (i),(ii), (iv) or (v). the kit comprising one or more polypeptides of (i) to(vi) and one or more test compounds.

The presently disclosed subject matter further provides an isolatedpolypeptide comprising the amino acid sequence as set out in SEQ IDNO:3, and an isolated nucleic acid encoding a polypeptide comprising theamino acid sequence as set out in SEQ ID NO:3. In certain embodiments,the isolated nucleic acid comprising the nucleotide sequence of SEQ IDNO:4.

The presently disclosed subject matter further provides a vectorcomprising the nucleic acids disclosed herein, and a host cellcontaining the polypeptides the nucleic acids, or the vectors disclosedherein.

The presently disclosed subject matter further provides a fusion proteincomprising a polypeptide comprising; (i) the sequence of a feline orcanine GPR120 or a feline or canine CD36 receptor; (ii) the amino acidsequence as set out in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ IDNO:7; (iii) an amino acid sequence having at least 90% identity to SEQID NO:1, SEQ ID NO:3; (iv) an amino acid sequence having at least 90%identity to SEQ ID NO: 5 or SEQ ID NO:7; (v) an amino acid sequencecomprising amino acids 127 to 279 of SEQ ID NO:3 or SEQ ID NO:7; (vi) afunctional fragment of (i), (ii), (iii), (iv) or (v).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of feline GPR120.

FIG. 2 shows the nucleotide sequence of feline GPR120.

FIG. 3 shows the amino acid sequence of feline CD36.

FIG. 4 shows the nucleotide sequence of feline CD36.

FIG. 5 shows the amino acid sequence of canine GPR120.

FIG. 6 shows the nucleotide sequence of canine GPR120.

FIG. 7 shows the amino acid sequence of canine CD36.

FIG. 8 shows the nucleotide sequence of canine CD36.

FIG. 9 shows the amino acid sequence of human GPR120.

FIG. 10 shows the nucleotide sequence of human GPR120.

FIG. 11 shows the amino acid sequence of human CD36.

FIG. 12 shows the nucleotide sequence of human CD36.

FIG. 13 shows the sequence difference between SEQ ID NO:3 and publishedfeline CD36 sequences.

FIG. 14 shows a feline dose response curve for oleic acid.

FIG. 15 shows a feline dose response curve for linoleic acid.

FIG. 16 shows a feline dose response curve for lauric acid.

FIG. 17 shows a feline dose response curve for palmitic acid.

FIG. 18 shows canine response curves for linoleic acid.

FIG. 19 shows canine response curves for oleic acid.

FIG. 20 shows the predicted structure of feline GPR120.

FIG. 21 shows the predicted structure of human CD36.

FIG. 22 shows feline GPR120 transient transfections in a stable cellline.

FIG. 23 shows feline GPR120 transient transfections in CHOK1 cells.

FIG. 24 shows free fatty acid dose response curves and EC₅₀ valuesobtained using an in vitro assay for feline GRP120.

FIG. 25 shows free fatty acid dose response curves and correspondingEC₅₀ values obtained using in vitro assay for feline GPR120 alone andco-transfected with CD36.

FIG. 26 shows fluorescence response for cat CD36 only in the presence ofSSO, an antagonist of CD36.

FIG. 27 shows a schematic of linoleic acid in the binding site of GPR120using an in silico method.

FIG. 28 shows a schematic of oleic acid in the binding site of GPR120using an in silico method.

DETAILED DESCRIPTION OF THE INVENTION

Therefore, in one aspect, the present invention provides a method foridentifying a compound that binds to and/or modulates the activity of apolypeptide, the polypeptide comprising;

-   -   (i) the sequence of a feline or canine GPR120 or a feline or        canine CD36 receptor;    -   (ii) the amino acid sequence as set out in SEQ ID NO:1, SEQ ID        NO:3, SEQ ID NO:5, SEQ ID NO:7;    -   (iii) an amino acid sequence having at least 90% identity to SEQ        ID NO:1 or SEQ ID NO:3;    -   (iv) an amino acid sequence having at least 90% identity to SEQ        ID NO:5 or SEQ ID NO:7;    -   (v) an amino acid sequence comprising amino acids 127 to 279 of        SEQ ID NO:3 or SEQ ID NO:7; or    -   (vi) a functional fragment of (i), (ii), (iii), (iv) or (v)    -   the method comprising determining whether a test compound binds        to and/or modulates the activity of the polypeptide.

The inventors have found that polypeptides comprising the sequence ofSEQ ID NO:1 and SEQ ID NO:3 are amino acid sequences of the felinehomologues of the human GPR120 and CD36 fatty acid receptors,respectively. SEQ ID NO:5 and SEQ ID NO:7 are the amino acid sequencesof the canine GPR120 and CD36 receptors, respectively. The humansequences are shown in SEQ ID NO:9 and SEQ ID NO:11, respectively.

The sequences were obtained from feline or canine re-sequenced genomicDNA and compared to human sequences and to suggested predicted felineand canine sequences. Some differences were found in the feline CD36gene sequence, between that published and that isolated by theinventors. Also included in the invention is the use of functional andallelic variants, which may differ in sequence but remain able to bestimulated by fatty acids, and lead to the perception of fatty acids bythe animal and as such, within a screening method.

The feline and canine sequences are predicted to be active, functionalreceptors, due to the sequence similarity to the human, rat and murineGPR120 and CD36 sequences that are available (at least 82% similarity).There is no reason not to believe that such receptors are not functionalin vivo, particularly in view of the fact that the inventors have showna clear response to feline and canine animals to oleic and linoleicacid, which are known to bind to the equivalent human receptors, and invitro assays show binding and responses of these receptors to knownligands.

Thus, in an aspect of the invention, the method is an in vitro method.The in vitro method may comprise:

-   -   Measuring the biological activity of the polypeptide in the        absence and in the presence of a test compound; and    -   Identifying an agent as one which binds to or modulates the        biological activity of the polypeptide, when there is a        difference between the biological activity in the absence        compared to the presence of the test agent.

The in vitro method may further comprise contacting the polypeptide witha test compound.

Detection methods for use in the method may include the use of alabelled compound/agent, and after washing determining which testcompounds remain bound to the receptors. Detecting activity induced bythe binding of a compound to the receptor may be by way of monitoringthe free calcium concentration within the cell which increases as aresult of receptor activation known as calcium flux, as well known to aperson skilled in the art. Monitoring may be by way of fluorescencedetection, such as a calcium sensitive fluorescent dye, or luminescencedetection, using a luminescent protein. An alternative method involvescGMP activity monitoring as also known by the skilled person.

The region between amino acid residues 127 and 279 of CD36 has beenimplicated in long chain fatty acid binding in humans, and thus, apolypeptide comprising this portion of SEQ ID NO:3 or SEQ ID NO:5 may beused in a method of the invention. A polypeptide comprising amino acidresidues 155 to 183 of SEQ ID NO:3 or SEQ ID NO:5 may be used in ascreening method of the invention.

The method may also involve the use of two polypeptides of the inventionat the same time, since the CD36 protein may act as a chaperone in orderto allow a compound or agent to interact with the protein of GPR120 orto increase the interaction between GPR120 and the fatty acid or otheractivating compound. Thus, an in vitro method comprising bothpolypeptides is included as a further aspect of the invention. As such,the invention includes a method comprising measuring the biologicalactivity of a GPR120 polypeptide (SEQ ID NO:1 or SEQ ID NO:5) or afragment thereof in the presence of a CD36 polypeptide (SEQ ID NO:3 ORSEQ ID NO:7 respectively) in the absence and in the presence of a testcompound; and identifying such an agent that causes a difference inactivity compared to the activity in the absence of the agent.

Methods of screening for agents which can modulate a biological activityof a polypeptide are well known in the art, and may involve the use ofsolid supports to which polypeptides of the invention are immobilised.

Agents identified by such screening methods may inhibit/antagonise oractivate/agonise the biological activity of a peptide of the invention.Thus, such agents may be useful as receptor agonists or antagonists.

Compounds identified by the in vitro method of the invention may befurther tested in vivo, for example, in feeding tests.

The invention also relates to a method for identifying a taste compoundthat binds to and/or modulates the activity of a GPR120 receptor and/ora CD36 receptor, wherein the GPR120 or CD36 receptor is feline, canineor human (SEQ ID NOs: 1, 3, 5, 7, 9 or 11) or wherein the GPR120receptor is at least 87% identical to SEQ ID NO:1, and wherein the CD36receptor is at least 82% identical to SEQ ID NO:3.

The GPR120 receptor may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% identical to any of SEQ ID NOs: 1, 5 or 9.

The CD36 receptor may be 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% to any of SEQ ID NOs: 3, 7 or 11.

The identification of such taste compounds may result in more palatablefoodstuff additives for cat, dog or human consumption.

It is desirable to identify compounds that are more beneficial thancompounds already known to bind to GPR120 and/or CD36; examples includecompounds that are easier/more cost effective to produce; compounds thatcan be used in smaller quantities for similar effects to knowncompounds; compounds that interact synergistically with other compounds.

The invention may also concern the receptors known as FFAR1 (GPR40),FFAR2 (GPR43) and/or FFAR3 (GPR41). These polypeptides have beendescribed previously as binding to fatty acids, but neither in thecontext of taste compounds, nor in the context of feline and canineanimals.

Methods as herein described for identifying compounds that bind toand/or modulate the biological activity of FFAR1, 2 or 3 receptors aretherefore also included within the scope of the invention.

All features of each aspect apply to each other aspect mutatis mutandis.

Amino acid sequences are described herein using the standard singleletter code. The sequences are described in the direction from theN-terminus to the C-terminus from left to right. The amino acids whichcan be incorporated into the peptides include any of the known naturallyoccurring amino acids.

In addition, the peptides of the invention may also include modifiedamino acids, that is, amino acids which do not naturally occur innature. For example, the peptides of the invention may includenorleucine, or other modified amino acids known in the art.

The peptides of the invention may consist only of the amino acidsequences disclosed herein, or may comprise other amino acids inaddition to those sequences. The polypeptide sequences described hereinmay contain additional amino acids at the N-terminal (the aminoterminal) end and/or at the C-terminal (the carboxy terminal) end of thesequences, particularly when used in a screening method of theinvention. Such additional amino acids may assist with immobilising thepolypeptide for screening purposes, or allow the polypeptide to be partof a fusion protein, for ease of detection of biological activity.

The polypeptides of the invention include homologues or derivatives ofthe above sequences, which retain the ability to bind medium to longchain fatty acids. A large number of conservative amino acidsubstitutions can be introduced into the peptide without causing anysignificant structural or functional changes. Thus, it may be possibleto replace one amino acid with another of similar “type”, for instance,replacing one hydrophobic amino acid with another. Suitable conservativeamino acid substitutions are known in the art. In the case of suchhomologues and derivatives, the degree of identity with the specificsequences identified herein is less important than that the homologue orderivative should retain the ability to bind a fatty acid and for asignal to be transmitted downstream. However, suitably, homologues orderivatives having at least 90% identity to the sequences providedherein are provided. Most preferably, homologues or derivatives havingat least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity areprovided.

The percent identity of two amino acid sequences or of two nucleic acidsequences is determined by aligning the sequences for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence for bestalignment with the sequence) and comparing the amino acid residues ornucleotides at corresponding positions. The “best alignment” is analignment of two sequences which results in the highest percentidentity. The percent identity is determined by the number of identicalamino acid residues or nucleotides in the sequences being compared(i.e., % identity=number of identical positions/total number ofpositions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and) (BLAST programsof Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilised as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilising BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g.,)(BLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.Another example of a mathematical algorithm utilised for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the CGC sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

Experiments with the human and rodent GPR120 and CD36 proteins have beencarried out with linoleic acid and oleic acid, many of them withknock-out rodent models. While these show the effect of lacking thereceptor they do not specifically show that the molecule activates thereceptor. An example showing oleic and linoleic acid responses in vitrofor Human GPR120 experiments in vitro is described in Galindo et al 2011Chem. Sen. Human CD36 experiments are described in (Kuda et al 2013 J.Biol. Chem), in relation to linoleic acid. Cartoni et al, 2010 J.Neurosci. showed that GPR120 knock-out mice had altered responses tolinoleic and oleic acid. Gaillard et al (2008, FASEB) showed that mouseCD36+ taste receptor cells were sensitive to linoleic acid while CD36−taste cells were not. The human and rodent homologues of thepolypeptides described herein have been shown to bind to these specificlong chain fatty acids. The feline and canine equivalent sequencesappear to bind to such molecules in view of the in vivo response tofatty acids at increasing concentrations, as shown herein. Furthermore,herein described in vitro assays with the feline receptors show apositive activation by linoleic and oleic acid.

The peptides for use in the screening methods of the invention may beproduced by chemical synthesis methods well known in the art. Forexample, the peptides may be synthesized chemically, using solid phasepeptide synthesis. These methods employ either solid or solution phasesynthesis methods (see for example, J. M. Stewart, and J. D. Young,Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., RockfordIll. (1984) and G. Barany and R. B. Merrifield, The Peptides: AnalysisSynthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 AcademicPress, New York, 1980, pp. 3-254 for solid phase synthesis techniques;and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis,Synthesis, Biology, supra, Vol 1, for classical solution synthesis).Other peptide synthesis methods are known in the art.

Alternatively, the peptides may be produced by expressing nucleic acidmolecules encoding precursors of the peptides. In another aspect, theinvention provides a nucleic acid sequence encoding a precursor of thepeptides of the invention. Such nucleic acids can be synthesised bymethods which are well known in the art (for example, see MolecularCloning: A Laboratory Manual: 3^(rd) Edition Sambrook and Russell, 2001,Cold Spring Harbor Laboratory Press).

In addition, peptide-encoding nucleic acids may be incorporated in asuitable nucleic acid vector. In a further aspect, the inventionprovides a vector comprising the nucleic acid of the invention. Thevector may have a promoter element operably linked to thepeptide-encoding nucleic acid sequence. Suitable vectors and methods ofproducing such vectors are known in the art.

The nucleic acid of the invention or vector of the invention may beintroduced into a host cell. Accordingly, in an additional aspect, theinvention provides a cell comprising the nucleic acid or vector of theinvention. The cell may be an isolated cell, such as a CHO K1 cell, orother suitably known stable cell line.

In a further aspect, the present invention provides fusion proteinsincluding the polypeptides described herein. Such fusion proteins maycontain a detectable marker, a functional group such as a carrier, alabel, a stabilising sequence or a mechanism by which fatty acid bindingmay be detected. Suitable labels include a FLAG tag, His tag, MYC tag, amaltose binding protein and others known in the art. The invention alsoprovides nucleic acids encoding such fusion proteins, vectors containingfusion protein-encoding nucleic acids, and host cells comprising suchnucleic acids or vectors.

Methods of synthesising such fusion proteins are well known in the art.

The method of the invention may be an in silico method. Such a methodmay comprise:

-   -   (i) predicting the (3-dimensional) 3D structure of the        polypeptide;    -   (ii) screening the predicted 3D structure of the polypeptide in        silico with a test compound;    -   (iii) predicting whether the test compound interacts with the        binding site of the polypeptide; and    -   (iv) identifying a compound as one that binds to and modulates        the biological activity of the polypeptide when the 3D structure        of the compound fits the binding site of the 3D structure of the        polypeptide.

Such techniques and methods are known in the art to the skilled person.Models of GPR120 were built using crystal structures of other Group AGPCRs as templates for homology modelling that were available from theProtein Data Bank, as would normally be performed by someone skilled inthe art. The Modeler software package was used. Simulations andminimizations for individual free fatty acids (e.g. linoleic acid) wereperformed, as would normally be performed by someone skilled in the art.Any suitable modelling software package may be used, as can suitablesimulation software programs.

A compound identified by the in silico screen of the invention asbinding to GPR120 or to CD36 may be further tested by the in vitromethod of the invention. Additionally or alternatively such a compoundmay be tested in vivo, for example, in feeding tests.

A further aspect of the invention provides compounds that modulate thebiological activity of a GPR120 receptor or a CD36 receptor, inaccordance with the first aspect of the invention.

The method of this aspect of the invention is therefore a suitablescreening method for identifying taste compounds that may be used in afoodstuff for a feline or canine animal to ensure long term acceptanceand consistent ingestion of such a foodstuff.

Thus, in an additional aspect, the invention provides a foodstuffcomprising an agent or compound identified by the method of theinvention.

The foodstuff may be any known in the art. A compound identified by themethod of the invention may be incorporated into any product which afeline or canine may consume in its diet. Thus, the invention coversstandard food products, supplements, pet food, drinks, snacks andtreats. The food product is preferably a cooked product. It mayincorporate meat or animal derived material (such as beef, chicken,turkey, lamb, blood plasma, marrowbone etc., or two or more thereof).The food product alternatively may be meat free (preferably including ameat substitute such as soya, maize gluten or a soya product) in orderto provide a protein source. The product may contain additional proteinsources such as soya protein concentrate, milk proteins, gluten etc. Theproduct may also contain a starch source, such as gelatinised starch,such as one or more grains (e.g. wheat, corn, rice, oats, barely etc.)or may be starch free. A typical dry commercial cat food contains about10-70% crude protein, about 10-60% fat and the remainder beingcarbohydrate, including dietary fibre and ash. A typical wet or moistproduct contains (on a dry matter basis) about 40% fat, 50% protein andthe remainder being fibre and ash. The present invention is particularlyrelevant for a pet foodstuff as herein described which is sold as adiet, foodstuff or supplement for a cat or a dog. In the present textthe term “domestic” cat mean cats, in particular Felis domesticus (Feliscatus) and the term “domestic” dog means dogs, in particular Canis lupusfamiliaris. Preferably, the pet foodstuff will meet the macronutrientrequirements of the animal.

Preferred features of each aspect of the invention are as for each ofthe other aspects, mutatis mutandis.

All referenced documents are disclosed herein by the fullest extentpermitted by law.

EXAMPLES

The invention will now be described with reference to the followingnon-limiting examples. Reference is made to the accompanying figures.

Example 1

Determining the Correct Sequence of Feline GPR120.

DNA was collected from 26 cats using cheek swabs. Two swabs werecollected from each cat. DNA extracted with the Qiagen DNeasy Blood andTissue Kit was used for sequencing. Primers were designed to flankexonic regions based on the publicly available feline genome sequence.All exonic regions were sequenced in both directions where possible.Sequences were analysed using Sequencher v5.1 (Gene Codes, USA).Consensus sequences from the 26 cats were compared with the publiclyavailable sequence and a final consensus sequence for all exons wasgenerated.

These sequences are based on re-sequenced WCPN cat data with referenceto RNA-Seq data and publicly available sequences for cat and human.

The confirmed fGPR120 coding sequence matches the sequence on Ensembl.This is the correct isoform as the other long isoform identified inhumans does not signal functionally.

Example 2

Determining the Correct Sequence of Feline CD36.

DNA was collected from 26 cats using cheek swabs. Two swabs werecollected from each cat. DNA extracted with the Qiagen DNeasy Blood andTissue Kit was used for sequencing. Primers were designed to flankexonic regions based on the publicly available feline genome sequence.All exonic regions were sequenced in both directions where possible.Sequences were analysed using Sequencher v5.1 (Gene Codes, USA).Consensus sequences from the 26 cats were compared with the publiclyavailable sequence and a final consensus sequence for all exons wasgenerated.

These sequences are based on re-sequenced WCPN cat data with referenceto RNA-Seq data, cDNA sequencing data from feline taste buds andpublicly available sequences for cat and human.

The transcript sequences available on Ensembl for both human and catcontain sections after the first stop codon. It is likely that in thecat, as is the case for human, the first portion of the transcriptsequence up to the first stop codon is the primary coding sequence. Atposition 300 there is a run of 8 adenine residues. This differs from thepredicted transcript on Ensembl but results in a 472 amino acid proteinwhich matches the length of the other isoform in cat and matches thelength of the human protein. Therefore neither of the transcriptspredicted on Ensembl match this sequence exactly but sequencing of cDNAfrom cat taste papillae shows that this is the correct transcriptconfiguration.

Example 3

Determining the Correct Sequence of Canine GPR120.

DNA was collected from 84 dogs by small volume blood sample. Wholegenome sequencing using the Illumina platform was performed on allsamples giving an average coverage of 15×. Data was mapped to thereference genome using Bowtie2. Regions of interest were extracted usingin-house Perl scripts. Exonic regions were identified and a finalconsensus sequence for all exons was generated.

These sequences are based on genome sequencing dog data with referenceto RNA-Seq data and publicly available sequences for dog and human.

The confirmed canine GPR120 coding sequence matches the sequence onEnsembl.

Example 4

Determining the Correct Sequence of Canine CD36.

DNA was collected from 84 dogs by small volume blood sample. Wholegenome sequencing using the Illumina platform was performed on allsamples giving an average coverage of 15×. Data was mapped to thereference genome using Bowtie2. Regions of interest were extracted usingin-house Perl scripts. Exonic regions were identified and a finalconsensus sequence for all exons was generated.

These sequences are based on genome sequencing dog data with referenceto RNA-Seq data and publicly available sequences for dog and human.

The confirmed canine CD36 coding sequence matches the sequence onEnsembl.

Example 5

Feeding Test to Determine Feline Response to Oleic Acid.

A cat gel panel was used to compare the palatability of a range ofconcentrations of oleic acid in a monadic exposure. The dose responsetested 8 concentrations of oleic acid ranging from 0.001% oleic acid to1% oleic acid. All products (including the blank, 0% oleic acid)contained 25 mM L-histidine as an ingestive/positive tastant to increasethe baseline gel intake, enabling the identification of a potentialnegative impact of the oleic acid concentration.

Oleic acid concentrations of 0.1%, 0.2%, 0.3%, and 0.6% (w/v) had asignificantly higher intake compared to the blank (0% oleic acid),showing that cats were able to taste the linoleic acid.

Example 6

Feeding Test to Determine Feline Response to Linoleic Acid.

A cat gel panel was used to compare the palatability of a range ofconcentrations of linoleic acid in a monadic exposure. The dose responsetested 8 concentrations of linoleic acid ranging from 0.001% linoleicacid to 1% linoleic acid. All products (including the blank, 0% linoleicacid) contained 25 mM L-histidine as an ingestive/positive tastant toincrease the baseline gel intake, enabling the identification of apotential negative impact of the linoleic acid concentration.

The linoleic acid concentration of 0.1% (w/v) had a significantly higherintake compared to the blank (0% linoleic acid). There was a trend forthe higher concentrations to become aversive/negative, with a reducedintake compared to the blank (0% linoleic acid), showing that cats wereable to taste the linoleic acid.

Example 7

Feeding Test to Determine Feline Response to Lauric Acid.

A cat gel panel was used to compare the palatability of a range ofconcentrations of lauric acid in a monadic exposure. The dose responsetested 5 concentrations of lauric acid ranging from 0.05% lauric acid to1% lauric acid. All products (including the blank, 0% lauric acid)contained 25 mM L-histidine as an ingestive/positive tastant to increasethe baseline gel intake, enabling the identification of a potentialnegative impact of the lauric acid concentration.

The lauric acid concentration of 0.1% had the highest intake overallcompared to the blank (0% lauric acid). However, the highestconcentrations tested of 0.6% and 1% lauric acid were significantlyaversive/negative compared to the blank (0% lauric acid), also showingthat cats were able to taste the lauric acid.

Example 8

Feeding Test to Determine Feline Response to Palmitic Acid.

A cat gel panel was used to compare the palatability of a range ofconcentrations of palmitic acid in a monadic exposure. The dose responsetested 5 concentrations of palmitic acid ranging from 0.05% palmiticacid to 1% palmitic acid. All products (including the blank, 0% palmiticacid) contained 25 mM L-histidine as an ingestive/positive tastant toincrease the baseline gel intake, enabling the identification of apotential negative impact of the palmitic acid concentration.

There was no significant difference in the intake of any of the palmiticacid concentrations tested, due to the fact that it is solid at roomtemperature (melting temperature approximately 63° C.). Therefore, thepalmitic acid was not able to interact and bind with the fatty acidreceptors to produce a taste response by the cats.

Example 9

Feeding Test to Determine Canine Response to Linoleic Acid.

Two different dog panels were used to compare the palatability ofconcentrations of linoleic acid. Each panel was made up of a singlebreed of dog; each panel being a different breed. The canine panels wereused to compare the palatability of a range of concentrations oflinoleic acid in a monadic exposure. The dose response tested 3concentrations of linoleic acid ranging from 0.01% linoleic acid to 1%linoleic acid. All products (including the blank, 0% linoleic acid)contained 100 mM L-histidine as an ingestive/positive tastant toincrease the baseline gel intake, enabling the identification of apotential negative impact of the linoleic acid concentration.

The different breeds demonstrated different responses to linoleic acid.Breed 1 had a significant positive/ingestive response for linoleic acidat the highest concentration tested of 1% compared to the blank (0%linoleic acid), while Breed 2 had smaller differences between thelinoleic acid concentrations tested.

Example 10

Feeding Test to Determine Canine Response to Oleic Acid.

Two different dog panels were used to compare the palatability ofconcentrations of oleic acid. Each panel was made up of a single breedof dog; each panel being a different breed.

The canine panels were used to compare the palatability of a range ofconcentrations of oleic acid in a monadic exposure. The dose responsetested 3 concentrations of oleic acid ranging from 0.01% oleic acid to1% oleic acid. All products (including the blank, 0% oleic acid)contained 100 mM L-histidine as an ingestive/positive tastant toincrease the baseline gel intake, enabling the identification of apotential negative impact of the oleic acid concentration.

The different breeds both demonstrated a response to oleic acid. Breed 1had a significant positive/ingestive response for oleic acid at thehighest concentration tested of 1% compared to the blank (0% oleicacid), while breed 2 had a significant positive/ingestive response foroleic acid at 0.1% compared to the blank (0% linoleic acid). This datashows that the dogs were able to taste the oleic acid.

Example 11

Method for GP120 and GPR120+CD36 Receptor In Vitro Assays Developmentand Use.

Initially the gene sequences for the target receptors GPR120 and CD36were confirmed by re-sequencing the genes of cats and dogs.

In the case of GPR120 (FFAR4, O3FAR1) the sequences obtained werecompared with the currently available feline or canine referencesequence and the human reference sequence. Sequences for the shortisoform were used.

In the case of CD36 the sequences obtained were compared with thecurrently available feline or canine reference sequence and the humanreference sequence.

Once target sequences were established they were synthesised and clonedinto the expression vectors pcDNA3.1Hygro and pcDNA3.1G418. Theseconstructs were then transiently transfected into the CHO K1immortalised cell line, and other commonly used cell lines usingLipofectamine 2000 and testing was performed to establish the successfulexpression of the target protein. The testing was carried out using acalcium sensitive fluorescent dye (Fluo8). Transfected cells were seededinto 384 well assay plates. By loading the cells with the dye and thenchallenging the cells with an agonist for the receptor the response ofthe cells could be recorded on the FLIPR^(TETRA) instrument by measuringthe increase in fluorescence associated with intra-cellular calciumrelease, thus confirming the functional expression of the receptor.Suitable experimental controls eliminate any possibility that theresponse of the cells is non-specific or that the fluorescence increaseis due to factors other than the release of intracellular calcium by thecells.

Both human and cat GPR120 showed specific responses to fatty acids inthe micro-molar range when transiently expressed in the stable cell lineA or CHOK1 cell line (FIG. 22 and FIG. 23, respectively). The humanreceptor did not require the presence of an exogenous G-protein but thecat receptor did require this in the stable cell line A. Dose responsecurves were generated for all the compounds tested (FIG. 24) and EC₅₀values were calculated. The effect of co-transfection of GPR120 and CD36is shown in FIG. 25.

Example 12

Method for CD36 Receptor In Vitro Assay Development and Use.

Initially the gene sequence for the target receptor CD36 was confirmedby re-sequencing the genes of cats and dogs.

The CD36 sequences obtained were compared with the currently availablefeline or canine reference sequence and the human reference sequence.

Once target sequence was established it was synthesised and cloned intothe expression vectors pcDNA3.1Hygro. The construct was then transientlytransfected into the CHO K1 immortalised cell line and other commonlyused cell lines using Lipofectamine 2000 and testing was performed toestablish the successful expression of the target protein. The testingwas carried out using a calcium sensitive fluorescent dye (Fluo8).Transfected cells were seeded into 384 well assay plates. By loading thecells with the dye and then challenging the cells with an agonist forthe receptor the response of the cells could be recorded on theFLIPR^(TETRA) instrument by measuring the increase in fluorescenceassociated with intra-cellular calcium release, thus confirming thefunctional expression of the receptor. Suitable experimental controlseliminate any possibility that the response of the cells is non-specificor that the fluorescence increase is due to factors other than therelease of intracellular calcium by the cells.

Further experiments with CD36 were performed to establish whether theputative CD36 antagonist Sulfo-N-succinimidyl Oleate (SSO) would inhibitCD36 mediated calcium influx after pre-treatment with Thapsigargin. Theresponse of cells transfected with CD36 or a mock control are shown inFIG. 28.

Example 13

Method for GPR120 Receptor in Silico Model Development and Use.

Models of GPR120 were built using crystal structures of Group A GPCRs astemplates for homology modelling that were available from the ProteinData Bank. The Modeler software package was used.

Simulations and minimizations for individual free fatty acids (e.g.linoleic acid) were performed. The program Charmm in Discovery Studiowas used.

What is claimed is:
 1. A method for increasing palatability of a petfoodstuff comprising: a. contacting a polypeptide with a compound,wherein the polypeptide comprises CD36 comprising the amino acidsequence set forth in SEQ ID NO:3 or SEQ ID NO:7, b. measuring thebiological activity of the polypeptide in the absence and in thepresence of the compound, and c. admixing the compound or a compositioncomprising the compound with a pet foodstuff when there is a differencebetween the biological activity in the absence, compared to the presenceof the compound.
 2. A method according to claim 1, wherein the method isan in vitro method.
 3. The method of claim 1, wherein the biologicalactivity of the polypeptide is measured in a cell comprising thepolypeptide.
 4. The method of claim 2, wherein the polypeptide isexpressed in a cell.
 5. The method of claim 4, wherein the polypeptideis expressed by a vector.
 6. The method of claim 4, wherein thebiological activity of the polypeptide is measured by monitoring acalcium concentration or a cGMP activity within the cell.
 7. The methodof claim 6, wherein the calcium concentration is monitored byfluorescence detection or luminescence detection.
 8. The method of claim7, wherein the fluorescence detection comprises a calcium sensitivefluorescent dye.
 9. The method of claim 1, further comprising testingthe compound in an animal feeding test.
 10. The method of claim 1,wherein the compound is present at a concentration of between 0.001% and1% in the pet foodstuff.
 11. The method of claim 1, wherein the compoundis present at a concentration of between 0.01% and 1% in the petfoodstuff.